Hematodinium Sp. Infection of Red Paralithodes Camtschaticus and Blue Paralithodes Platypus King Crabs from the Sea of Okhotsk, Russia ⇑ T.V

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Journal of Invertebrate Pathology 105 (2010) 329–334

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Journal of Invertebrate Pathology

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Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia ⇑ T.V. Ryazanova a, M.G. Eliseikina b, A.D. Kukhlevsky b, V.I. Kharlamenko b, a Kamchatka Research Institute of Fisheries and Oceanography, Petropavlovsk-Kamchatsky 683002, Russia b A.V. Zhirmunsky Institute of Marine Biology FEB RAS, Palchevsky str. 17, Vladivostok 690041, Russia article info abstract

Article history: A disease caused by a parasitic dinoflagellate of the genus Hematodinium was identified in red, Paralithodes Received 8 December 2009 camtschaticus, and blue, Paralithodes platypus, king crabs from the north-east region of the Sea of Okhotsk, Accepted 29 July 2010 Russia, during annual stock surveys. No carapace color change was observed even in heavily infected crabs, Available online 5 August 2010 but diseased crabs possessed creamy-yellow hemolymph, which was visible through the arthrodial membranes of the abdomen and appendages. Several stages of the parasite’s life history, including trophonts, Keywords: plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. Numerous par- Hematodinium sp. asite cells were observed in the lumina of the myocardium, the gills, the connective tissue of antennal Paralithodes camtschaticus glands and the sinuses of nerve ganglia, eyestalks and gastrointestinal tract of king crabs with gross signs Paralithodes platypus Sea of Okhotsk of infection. Based on sequencing of the 18S rDNA, it appears that the Hematodinium sp. found in red and Infection blue king crabs is identical or closely related to Hematodinium sp. isolated from crabs of the genera Chion- Prevalence oecetes and Lithodes. Observed prevalences were 0.33% in sublegal male red king crabs, 0.18% in female red Parasitic dinoflagellate king crabs, 0.34% in sublegal male blue king crabs and 0.31% in female blue king crabs. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction crab ( Chionoecetes bairdi) hemolymph developed detectable infections; and hemolymph smears from southeast Alaska crabs, The red king crab, Paralithodes camtschaticus, followed by the P. camtschaticus, P. platypus, and Lithodes aequispina contained no blue king crab, Paralithodes platypus, are the most commercially dinoflagellate forms (Meyers et al., 1987). The first incident of valuable crab species in the Sea of Okhotsk, Russia. Combined an- Hematodinium sp. infection was observed on the West Kamchatka nual landings of these species total 50,000 metric tons and over re- shelf in 2002 in snow crabs, Chionoecetes opilio, and this disease cent decades, these landings have been stable when compared to was found in red and blue king crabs, P. platypus, from this area other regions (Otto and Jamieson, 2001). However, since 1999, four years later. the Sea of Okhotsk king crab stock has declined significantly and This report describes the results of gross or macroscopic exam- remains low (Dolgenkov and Koblikov, 2009). In addition to overf- ination as well as microscopic (light and transmission electron ishing, a number of other factors may impact crab population microscopy) observations of the infection in king crabs P. camtsch- structure, including diseases. In particular, infections caused by aticus and P. platypus, molecular identification of Hematodinium sp. the parasitic dinoflagellate Hematodinium sp., may significantly in king crabs, and it also includes information on the distribution of change the size and structure of important crab populations (Sten- infected crabs in the north-east region Sea of Okhotsk. tiford and Shields, 2005). Hematodinium sp. infections in various marine crustacean spe- 2. Materials and methods cies have been recorded in many areas of the world. Hematodini- um-associated diseases are generally fatal to the host (Messick Lithodid crabs, the red king crab P. camtschaticus and blue king and Shields, 2000), and its occurrence can reach 100% for some crab P. platypus, were used as material for the present research. The crustacean species (Messick, 1994). The majority of infections are survey took place in the northeastern area of the Sea of Okhotsk reported in brachyuran crabs (Stentiford and Shields, 2005). In during annual stock assessment surveys (Fig. 1). Crab trap and disease transmission studies, Meyers et al. (1987) reported that trawl surveys were used. The crab trap survey onboard the RV none of the lithodid king crabs inoculated with infected Tanner Ametist from August 25 to December 15 2006 was conducted from the north to the south. Traps were deployed in ‘fleets’ of 100 stan- ⇑ Corresponding author. dard Japanese conical traps baited with 1 kg of frozen herring. E-mail address: [email protected] (V.I. Kharlamenko). Eighty-eight fleets of traps were set between 64 and 322 m depth

drated in acetone and embedded in Epon-Araldite. Semithin and ultrathin sections were cut using a Leica EM UC6 ultramicrotome. Semithin sections were stained with methylene blue and examined using a Leica DM 4500 microscope. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Zeiss Libra 120 transmission electron microscope. The standard method for total DNA isolation from the tissue of crabs was used (Sambrook et al., 1989). Hematodinium-specific primers Hemat-F-1487 and Hemat-R-1654 were used to detect Hematodinium (Gruebl et al., 2002). PCR was performed using a GenAMP 9600 thermal cycler system (Perkin Elmer). Amplification conditions for the PCR included an initial denaturation step (94 °C for 10 min) and 30 amplification cycles (denaturation, 15 s at 94 °C; annealing, 15 s at 56 °C; elongation, 30 s at 72 °C). Reaction products were checked for size and purity on 1% agarose gels. To confirm the identity of the parasites detected in king crabs, repre- sentative 196 bp PCR amplicons were used as templates for sequencing amplification using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Purified sequencing products were analyzed by electrophoresis on a 50 cm capillary array of an ABI Prism 3130 DNA sequencer. Sequences were assembled with SeqScape v2.5 software (Applied Biosystems). In addition, a long segment of 18S rDNA (1682 bp) from the parasite detected in the red king crab was sequenced. Direct sequence determination of each of the obtained amplicons yielded a 1464 bp sequence. The consensus sequences were compared with homologous Hematodi- nium 18S rDNA sequences available in GeneBank. Calculation of similarity values was conducted using the program MEGA version 4(Tamura et al., 2007). The density of infected crabs was estimated with the program Chartmaster version 3.1 using the kriging interpolation method.

Fig. 1. Map of the study area with sampling stations.

3. Results at randomly selected locations in the range of latitudes from 58°510Nto57°10N and from 55°410Nto54°150N. The standard soak Overall, 11882 red king crabs and 4790 blue king crabs were time for traps was 36–48 h, but in some cases varied from 35 to subjected to gross examination. Gross signs of infection were 190 h depending on weather conditions. The 2007 trawl survey found in 14 red king crabs and nine blue king crabs. All infected was conducted onboard the RV Professor Kaganovsky from July 8 crabs were females or sublegal males (with the carapace width to August 1 beginning in the south and proceeding north. 146 from 75 to 102 mm) in the third intermolt stage (carapace and che- trawls from 51°050Nto57°420N were carried out within depth la are hard and cannot be depressed by thumb). No external range 14–200 m. The trawl sections were performed in a standard wounds were visible. None of the collected crabs changed color pattern at a speed of 3 knots for 30 min, using a DT 27.1 trawl. of their carapace, but infected crabs did possess a creamy-yellow All crabs were removed from the catch, sorted by species and hemolymph, which was visible through the arthrodial membranes sex. Crab size is reported as carapace width excluding spines. Crabs of abdomen and appendages (Fig. 2). Their dissection revealed that were divided into three groups – legal males (carapace width (cw) the hemolymph of suspect crabs appeared as a creamy-yellow fluid greater than or equal to 150 mm and 130 mm for red and blue mass surrounding the muscles of appendages and all internal or- kings crabs, respectively), sublegal (cw < 150 and 130 mm) for gans, filling the pericardial cavity and gill lamellae and stems. males and females, respectively. Intermolt state of the carapace The cooked meat of suspect crabs had a bitter and astringent taste. was assessed and assigned to one of six classes according to spe- Prevalence was 0.33%, 0.18%, 0.34% and 0.31% in sublegal male and cific criteria (Lysenko, 2001). In total, 3189 males and 1601 females legal female red king crabs, and legal female and sublegal male of the blue king crab, and 8587 males and 3295 females of the red blue king crabs, respectively. Gross evidence of infections was king crab were examined macroscopically. Randomly selected not found in legal male red or blue king crabs (Table 1). Although crabs with external signs of disease and additional individuals that the data are limited, prevalence of infection was different in trawl appeared healthy upon gross examination were dissected (700 and pot surveys. Infected crabs were found at depths ranging from males, 282 females of the red king crab, 230 males and 147 females to 30 to134 m. Although small in number, the majority of infected of the blue crab). From these, 77 red king crabs and 56 blue king crabs were located between latitudes 57°000N and 57°350N along crabs were selected for histological examination. Tissue samples the western Kamchatka shelf (Fig. 1). Interpolation suggests that were fixed for 24–48 h in Davidson’s fixative (Bell and Lightner, maximum density of infected red and blue king crabs was 84 1988) prepared in sea water and processed using standard histo- and 108 crabs per km2, respectively. In this area infection preva- logical techniques. Five-micron sections were stained with Meyer’s lence as determined by macroscopic signs was 0.61% in sublegal hematoxylin–eosin (H&E) and examined using Olympus Al-2 or male red king crabs, 0.83% in female red king crabs, 0.50% in sub- Leica DM 4500 light microscopes with an automatic camera. Seven legal male blue king crabs and 0.81% in female blue king crabs. infected crabs were studied by TEM. Tissue samples for transmis- Histopathological changes were similar in both crab species sion electron microscopy were fixed with 2.5% glutaraldehyde in with gross signs of infection and all infections appeared advanced, sterile sea water, post-fixed with 1% of 0s04 in sea water, dehy- with large numbers of parasite cells distributed throughout the T.V. Ryazanova et al. / Journal of Invertebrate Pathology 105 (2010) 329–334 331

Fig. 2. The sublegal males of king crabs infected by Hematodinium sp. (A) The cream-yellow hemolymph (arrow) is visible through the translucent covers of abdomen and appendage joints of blue king crab. (B) The internal organs of a red king crab surrounded by cream-yellow dense hemolymph.

Table 1 Prevalence of visually diagnosed Hematodinium sp. infections in king crabs from north-east region of the Sea of Okhotsk.

Paralithodes camtschaticus Paralithodes platypus Legal males Sublegal males Females Legal males Sublegal males Females Total number of crab examined Trawls 4722 1091 2934 153 51 312 Traps 1414 1360 361 1843 1142 1289 Total 6136 2451 3295 1996 1193 1601 Number of crabs infected Trawls 0 5 6 0 0 4 Traps 0 3 0 0 4 1 Total 0 8 6 0 4 5 Prevalence (%) Trawls 0 0.46 0.20 0 0.00 1.28 Traps 0 0.22 0.00 0 0.35 0.08 Total 0 0.33 0.18 0 0.34 0.31

tissues. Rounded trophonts (10.7 ± 1.7 lm) with distinct dinokary- creatic tubules, the wall of the gut and blood vessels. The otic nuclei and rounded or vermiform plasmodia, containing from connective tissue between the muscle fibrils was reduced, and 2 to 8 or more nuclei were found in 21 of 23 crabs (Fig. 3A). Three the sarcolemma was breached by the parasite cells (Fig. 4C). Some red king crabs had some plasmodia of vermiform shape with 2–5 small melanized nodules in the antennal gland, stomach and nuclei located in a row (Fig. 3B). hematopoietic tissue were found in three individuals. In crabs with advanced infections, identified by high densities of In the tissues of two of the more heavily infected crabs, the par- trophonts and plasmodia throughout, numerous parasite cells asite cells were smaller in size compared to the normal trophonts were observed in the lumen of the myocardium (Fig. 4A), the gill and their cytoplasm had a very small volume (Figs. 3C and 4D). The and connective tissue of the antennal gland (Fig. 4B), nerve ganglia, majority of the cells were uninucleated, but binucleated and other eyestalks, gastrointestinal tract, and testes, as well as between oo- multi-nucleated cells were also observed. The size of those cells cytes of females. The hematopoietic tissue of infected animals was was 4.45 ± 0.4 lm (mean ± standard deviation, N = 20), and the mitotically active, however few hemocytes were observed in circu- size of their nuclei was 3.35 ± 0.4 lm(N = 20). The nuclei of the lation. The hemal sinuses of the hepatopancreas were dilated and cells were filled with a granular nucleoplasm along with the poly- filled by large number of parasite cells, and the connective tissue morphous electron-dense heterochromatin clumps. The multive- between the tubules was replaced by these cells. Vermiform plas- sicular bodies and vacuoles with homogeneous contents were modial stages were attached to the basal membrane of hepatopan- located in the granular cytoplasm. Nuclear chromatin was dense

Fig. 3. Stages of Hematodinium sp. in king crabs. (A) Semithin section of trophonts and multinucleate plasmodial stages of Hematodinium sp. (methylene blue staining (scale bar = 50 lm)). (B) The vermiform plasmodia attached to the wall of a blood vessel (scale bar = 50 lm). (C) Pre-spore stage (scale bar = 50 lm). (D) Nomarski differential interference contrast microscopy showing the round trophont and goblet-like form cell with beak-like protrusion in ﬁxed hemolymph of sublegal male blue crab (scale bar = 20 lm). 332 T.V. Ryazanova et al. / Journal of Invertebrate Pathology 105 (2010) 329–334

Fig. 4. Light micrographs tissues from king crabs infected by Hematodinium sp. (A) The trophonts and plasmodium in the myocardium. (B) The parasite in the connective tissue of the antennal gland. (C) The parasite around and inside the muscle fibrils. (D) Transverse section of hepatopancreatic tubules showing replacement of the interstitial connective tissue and hemal sinuses by numerous parasites. Scale bar = 50 lm. and V-shaped. In the cytoplasm of the polynuclear and mononu- recovered from the red king crab was deposited in Genbank clear cells, the electron-dense trichocysts of a rhomb or a square (Accession Number EU856716) and showed 100% base pair simi- shape were observed with TEM (Fig. 5A and B). Using Nomarski dif- larity to the sequenced Hematodinium sp. (Genbank Accession ferential interference contrast microscopy, some goblet-like cells, Numbers FJ844412-FJ844431) isolated from L. couesi, Hyas coarct- having beak-like and keel-like protrusions, were found in the atus, C. bairdi, C. opilio, C. tanneri, C. angulatus, Callinectes sapidus hemolymph of one male blue crab (Fig. 3D). More severe patholog- and Nephrops norvegicus. This implies the presence of Hematodini- ical changes were observed in the tissue of two heavily infected um sp. in the tissues of red king crab. crabs. The connective tissue of all organs was replaced by parasites. The hemal sinuses of the hepatopancreas were dilated and oc- 4. Discussion cluded by large numbers of parasites, although few parasite cells were observed above the basement membrane (Fig. 4D). The tu- In the advanced stages of the disease, most species of crabs in- bules were often destroyed, and only basal membranes remained. fected with Hematodinium spp. develop changes in carapace color Muscle tissue was surrounded by parasites, nuclei of muscle fibers (Stentiford and Shields, 2005), and these changes are used as mac- were absent, and the fibril striation was lost. Muscle fibers had roscopic criteria for diagnosing Hematodinium spp. infection. The been almost completely replaced by the proliferating parasites sternae and ventral surfaces of the Australian sand crabs Portunus and only small islands of muscle fibrils were preserved. pelagicus had a chalky, white appearance (Hudson and Shields, Small 18S rDNA fragments amplified from red king crab had a 1994). The abdomen of dying velvet swimming crabs Necora puber 100% sequence similarity to respective gene fragments of the from several areas in France and Spain had a pale-pink color (Wil- Hematodinium sp. found in Lithodes couesi and four species of the helm and Mialhe, 1996). Changes in carapace color of some species genus Chionoecetes (Genbank Accession Numbers FJ844413, of crabs are even more noticeable. The edible crab Cancer pagurus FJ844414, FJ844418, FJ844423 and FJ844426). Based on this com- from UK waters exhibited an altered coloration (pink hyperpig- parison, the red king crab parasite was identified as Hematodinium mentation), and this infection is called ‘‘Pink Crab Disease” (Stenti- sp., and the sequence recovered from red king crab was deposited ford et al., 2002). The primary macroscopic sign of Hematodinium in Genbank (Accession Number EU856717). To confirm that the spp. infection in snow crabs, C. opilio, and Tanner crabs, C. bairdi, small 196 bp 18S rDNA PCR amplicon diagnostic for Hematodinium was a distinct change in carapace color that resulted in a cooked sp. was indeed derived from Hematodinium sp., 18S rDNA large appearance (Meyers et al., 1987), and this feature was used for gene fragments were amplified from three red king crabs. All three macroscopic estimation of disease prevalence (Meyers et al., 1464 bp sequenced amplicons were identical. The sequence 1990; Pestal et al., 2003). For red and blue king crabs in Kamchatka

Fig. 5. Transmission electron micrographs of Hematodinium sp. cells with trichocysts in cytoplasm. (A) Bi-nucleate parasitic cell (scale bar = 5 lm). (B) Uninucleate parasitic cell (scale bar = 2 lm). N: nucleus; mb: multivesicular body; t: trichocysts. T.V. Ryazanova et al. / Journal of Invertebrate Pathology 105 (2010) 329–334 333 waters, we did not register any color change even in highly in- missed. The accuracy of macroscopic diagnosis based on carapace fected crabs except for a creamy-yellow color of the hemolymph, color of C. opilio was around 0.53 (Pestal et al., 2003), and gross visible through the arthrodial membranes of the abdomen and inspection based on hemolymph changes is less accurate. For more appendages. Dissection showed that the hemolymph of the in- precise assessment it is necessary to use other methods, such as fected king crabs is creamy-yellow in color and did not clot. Cream microscopic analysis of hemolymph or molecular methods (see color and dense consistency of hemolymph in crabs in late stages Jensen et al., 2010). Infection of crustaceans with the Hematodini- of disease, such as registered in heavily infected king crabs, have um spp. dinoflagellates has a strongly pronounced seasonal pattern been described in many species of crustaceans infected with (Stentiford and Shields, 2005). The occurrence of the disease in the Hematodinium spp.(Meyers et al., 1987; Shields, 1994). In addition, Tanner crab C. bairdi in Alaska decreased in the autumn, as infected infected Tanner crabs have a bitter, astringent taste of cooked meat crabs died by that time (Love et al., 1993). In our study, gross signs (Messick, 1994; Wilhelm and Mialhe, 1996; Stentiford et al., 2002). of infection were absent in king crabs caught starting from the sec- Several stages of the parasite, including trophonts, plasmodia, ond half of October. sporonts and macrodinospores, were observed in tissues of in- Hematodinium infections have been reported in many brachyu- fected king crabs. The presence of trophonts and multinucleated ran crabs (Stentiford and Shields, 2005) and in the present study plasmodial stages of the parasite in internal organs of crustaceans we described this infection in anomuran crabs using a diverse is a major diagnostic attribute of Hematodinium sp. infection approach. (Meyers et al., 1987; Messick, 1994). Unattached vermiform plasmodia probably represent the motile form of the parasite, but Acknowledgments the use of only fixed material did not allow us to confirm this sug- gestion. Vermiform plasmodia have been observed in many deca- We thank the anonymous reviewers for valuable comments and pods (Stentiford and Shields, 2005), but only three king crabs suggestions to this manuscript. This work was supported, in part, with gross signs of infection had vermiform plasmodia. The reason by FEB RAS (Project 09-III-A-06-210). for the low prevalence of crabs with such plasmodia may be the relatively short duration of this stage (Appleton and Vickerman, 1998). Trichocysts were present in the parasites from heavily in- References fected king crabs. Studies of stages of Hematodinium sp. develop- Appleton, P.L., Vickerman, K., 1998. 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